Mechanistic Study of HCV Polymerase Inhibitors at Individual Steps of the Polymerization Reaction

Liu, Yaya; Jiang, Wen; Pratt, John K.; Rockway, Todd W.; Harris, Kevin S.; Vasavanonda, Sudthida; Tripathi, Rakesh; Pithawalla, Ron; Kati, Warren M.

doi:10.1021/bi060511j

Cited by 44 publications

(39 citation statements)

References 41 publications

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“…We tested RNAs derived from both isolates to exclude subtle differences in binding affinity due to potential isolate-specific cis-acting signals. Titration with increasing concentrations of RNA revealed a stoichiometry of only about 0.05 for the protein-RNA complex (data not shown), which compares to previously reported low values (20% [25]). The possibility of multiple NS5B molecules on the biosensor surface binding to the same RNA strand might account for the lower binding capacity determined here.…”

Section: Resultscontrasting

confidence: 61%

Structural and Functional Analysis of Hepatitis C Virus Strain JFH1 Polymerase

Simister

Schmitt

Geitmann

et al. 2009

J Virol

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The hepatitis C virus (HCV) isolate JFH1 represents the only cloned wild-type sequence capable of efficient replication in cell culture, as well as in chimpanzees. Previous reports have pointed to the viral polymerase NS5B as a major determinant for efficient replication of this isolate. To understand the underlying mechanisms, we expressed and purified NS5B of JFH1 and of the closely related isolate J6, which replicates below the limit of detection in cell culture. The JFH1 enzyme exhibited a 5-to 10-fold-higher specific activity in vitro, consistent with the polymerase activity itself contributing to efficient replication of JFH1. The higher in vitro activity of the JFH1 enzyme was not due to increased RNA binding, elongation rate, or processivity of the polymerase but to higher initiation efficiency. By using homopolymeric and heteropolymeric templates, we found that purified JFH1 NS5B was significantly more efficient in de novo initiation of RNA synthesis than the J6 counterpart, particularly at low GTP concentrations, probably representing an important prerequisite for the rapid replication kinetics of JFH1. Furthermore, we solved the crystal structure of JFH1 NS5B, which displays a very closed conformation that is expected to facilitate de novo initiation. Structural analysis shows that this closed conformation is stabilized by a sprinkle of substitutions that together promote extra hydrophobic interactions between the subdomains "thumb" and "fingers." These analyses provide deeper insights into the initiation of HCV RNA synthesis and might help to establish more efficient cell culture models for HCV using alternative isolates.Hepatitis C virus (HCV) is an enveloped positive-strand RNA virus belonging to the genus Hepacivirus in the family Flaviviridae. The genome of HCV encompasses a single ϳ9,600-nucleotide (nt)-long RNA molecule carrying one large open reading frame flanked by nontranslated regions (NTRs), which is primarily translated into one polyprotein. The polyprotein precursor is cleaved by cellular and viral proteases into at least 10 different products (for a review, see reference 5). The nonstructural proteins NS3 to NS5B are necessary and sufficient for autonomous RNA replication. They form a membraneassociated replication complex in which NS5B is the RNAdependent RNA polymerase (RdRp), the key enzyme of viral-RNA replication.Studies of HCV have long been hampered by the lack of efficient cell culture systems. The advent of subgenomic replicons derived from genotype 1 isolates allowed for the first time a concise analysis of authentic viral-RNA replication (29).However, these isolates required adaptive mutations for efficient propagation in cells (9,21,27). Based on these adapted viral sequences, it was not possible to generate infectious virus, since most of these mutations seemed to interfere with virus assembly (38), and only one cell culture-adapted genotype 1 isolate has yet given rise to moderately efficient production of infectious virus (49). This restriction was abolished by a particu...

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Section: Resultscontrasting

confidence: 61%

Structural and Functional Analysis of Hepatitis C Virus Strain JFH1 Polymerase

Simister

Schmitt

Geitmann

et al. 2009

J Virol

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“…It has previously been suggested that finger-loop inhibitors form either unproductive complexes or interfere with the conformational changes required to adopt a more open conformation (11)(12)(13)(14). Our data show that a prototype compound of this class reduces the shift in average FRET values (reduced binding) and FRET fluctuations (reduced sliding-wrapping dynamics) in a dose-dependent manner.…”

Section: Discussionsupporting

confidence: 50%

“…Thumb pocket 1 non-nucleoside inhibitors (NNIs) or finger loop NNIs bind to a region of the thumb that is otherwise occupied by the flexible ⌬1 finger loop, which seems to impede productive RNA binding (Fig. 1b) (11)(12)(13)(14).…”

Section: The Hepatitis C Virus (Hcv)mentioning

confidence: 99%

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Dynamics of Hepatitis C Virus (HCV) RNA-dependent RNA Polymerase NS5B in Complex with RNA

Karam

Powdrill

Liu

et al. 2014

Journal of Biological Chemistry

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Background:The dynamics associated with RNA binding by the hepatitis C virus (HCV) polymerase remain elusive. Results: Single molecule experiments reveal changing populations of binary RNA-enzyme complexes. Conclusion: Rapid enzyme conformational changes facilitate sliding/wrapping of the polymerase along its RNA substrate. Significance: This study provides novel insight into mechanisms associated with viral replication and its inhibition.

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“…Noncompetitive inhibition of NS5B polymerase activity with respect to NTPs has been reported (2, 15, 16). Based on co-crystallization studies with NS5B, it has been proposed that allosteric inhibitors may lock the NS5B protein in an inactive formation by binding tightly to the protein (16,17). It is important to understand how the binding affinity relates to inhibition potency and resistance to HCV inhibition.…”

Section: Hepatitis C Virus (Hcv)mentioning

confidence: 99%

Slow Binding Inhibition and Mechanism of Resistance of Non-nucleoside Polymerase Inhibitors of Hepatitis C Virus

Hang

Yang

Harris

et al. 2009

Journal of Biological Chemistry

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Hepatitis C virus (HCV)4 constitutes a global health problem. Current therapies are unable to effectively eliminate viral infection in a significant number of patients. The RNA-dependent RNA polymerase (RdRp) of HCV NS5B is an attractive target for the development of orally bioavailable small molecule inhibitors (1, 2). The structure of the NS5B apoenzyme and the NS5B-RNA complex reveals the characteristic right hand architecture of polymerase enzymes, comprising three distinct domains (palm, thumb, and finger) encircling the enzyme active site located in the palm domain (3-6). The structural and biochemical characterization of HCV NS5B polymerase can provide a basis for drug design efforts, and the elucidation of the mechanism of inhibition can guide the optimization of inhibitor efficiency against wild-type and resistant mutants.Among the extensively investigated non-nucleosides documented to inhibit the RdRp activity of HCV NS5B, derivatives of various benzofuran and benzothiadiazine have been reported to bind to allosteric binding sites in the palm domain of NS5B (7,8). The palm domain, whose geometry is conserved in virtually all DNA and RNA polymerases, contains catalytic aspartic acids responsible for the nucleotidyl transfer reaction. The benzofuran compound HCV-796 has been shown to have significant antiviral effects in patients chronically infected with HCV (9, 10). In addition, two series of compounds based on the thiophene and benzimidazole scaffolds have been reported to inhibit NS5B by binding to two different binding pockets in the thumb domain of NS5B (11,12). The thumb domain is connected to the palm domain by a ␤-hairpin termed the primer grip motif. The C-terminal region of the thumb protrudes toward the active site (3). The thumb binding inhibitors have been proposed to inhibit the RdRp activity of NS5B, perhaps by interfering with template/primer interaction and conformational dynamics of the protein (13,14).Despite the elucidation of a number of NNIs that bind to the thumb and palm binding sites, the mechanism by which NNIs cause inhibition of RNA synthesis is unclear. Also, our understanding of the kinetics of NNI interaction with NS5B, the role of NNI binding and kinetics for inhibition, and the inhibitor efficacy on NS5B-resistant mutations remains incomplete. The four representative palm-and thumb-binding NNIs selected in this study have been reported to effectively inhibit replication of subgenomic replicons with low toxicity. Noncompetitive inhibition of NS5B polymerase activity with respect to NTPs has been reported (2, 15, 16). Based on co-crystallization studies with NS5B, it has been proposed that allosteric inhibitors may lock the NS5B protein in an inactive formation by binding tightly to the protein (16,17). It is important to understand how the binding affinity relates to inhibition potency and resistance to HCV inhibition. Because the intrinsic potency of slowly binding compounds can be underestimated in the short time □ S The on-line version of this article (available at htt...

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Mechanistic Study of HCV Polymerase Inhibitors at Individual Steps of the Polymerization Reaction

Cited by 44 publications

References 41 publications

Structural and Functional Analysis of Hepatitis C Virus Strain JFH1 Polymerase

Structural and Functional Analysis of Hepatitis C Virus Strain JFH1 Polymerase

Dynamics of Hepatitis C Virus (HCV) RNA-dependent RNA Polymerase NS5B in Complex with RNA

Slow Binding Inhibition and Mechanism of Resistance of Non-nucleoside Polymerase Inhibitors of Hepatitis C Virus

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